INTRODUCTION
Japanese Brown (JBR) cattle, compared to the Japanese Black (JBL) breed, are a minor population breed with moderately marbled beef and high growth performance [
1]. In 2021, approximately 22,400 animals were reared in Japan. To date, the genetic background [
2], growth performance [
3], carcass [
4,
5] and beef characteristics of JBR cattle have been investigated [
6,
7]. This breed comprises two pedigrees, 10% animals of which are the Kochi (Tosa; JBRT), and the remainder are the Kumamoto [
8]. In recent years, JBR beef has been accepted by health conscious consumers owing to a balanced composition of lean muscle and intramuscular fat [
9]. Despite the increasing demand for this beef in the market, the details of its skeletal muscle properties remain poorly understood. The crude fat content in loin muscle was 26.6% on average (n = 45) in another JBR cattle (Kumamoto pedigree), whereas the content was 39.7% in JBL cattle, the original Wagyu breed with highly marbled beef [
1]. In addition, levels of carnosine, taurine, glutamine, and alanine were 9.63, 6.70, 5.21, and 3.71 μmol/g, respectively, in the loin of JBR Kumamoto cattle, whereas those in JBL cattle were 6.90, 5.33, 3.64, and 2.95 μmol/g, respectively. Total peptide content in the JBR and JBL cattle was 4.78 and 2.49 mg/g, respectively [
1]. These results suggest that the levels of flavor-related metabolites in the loin muscle are higher in JBR cattle than in JBL cattle.
In farm animals, metabolites play physiologically essential roles in skeletal muscles and contribute to nutritional value and palatability after death [
10]. During postmortem aging of porcine and bovine meat, proteins, glycogen, sugars, and energy-charged metabolites, such as adenosine triphosphate (ATP), are degraded through biochemical reactions [
11–
18]. An appropriate process of postmortem meat aging leads to preferable meat flavor development by increasing metabolites, not only amino acids (AAs) but also nucleotide degradation products such as inosine 5′-monophosphate (IMP) as flavor and taste components [
11,
19,
20]. In addition, these metabolites contribute to formation of “meaty aroma” as precursors of the flavor-related compounds [
21]. In particular, free AAs and peptides react with sugar-related metabolites via the Maillard reaction in meat during cooking [
22]. These compounds can be further converted via autooxidation and evaporated as volatile flavor compounds, such as 2,5-dimethyl-4-hydroxy-3(2H)-furanone (furaneol) [
23]. Thus, JBR beef may have a characteristic meat flavor that is stronger than that of JBL beef.
To understand the mechanisms underlying metabolite changes in such processes and conditions of meat production, metabolomic approaches have been employed in studies on postmortem meat aging [
12,
24–
29]. Recently, we demonstrated that postmortem metabolic changes progress in the
longissimus thoracis (LT) muscle metabolism of JBL cattle, including protein digestion, glycolysis, citric acid cycle, pentose phosphate pathway, and the metabolism of nicotinamide, purine, and glutathione [
24]. In addition, metabolomic profiles are affected by the genetic background of the breed in animals, and therefore, it is expected that metabolomic approaches can identify breed-specific muscle metabolites that characterize the meat [
30,
31]. Taking the interbreed difference into account, this led us to hypothesize that, in JBR cattle, skeletal muscle metabolism and the metabolites are different from those in JBL cattle. However, to date, interbreed differences in the metabolomes of bovine skeletal muscle and beef products have been poorly addressed. In particular, little is known about JBR muscle metabolites and their differences from those of JBL cattle, which may have an impact on interbreed differences in beef flavor.
To address this, we aimed to capture the metabolome profiles in the lean portion of LT muscle in JBR cattle to characterize the beef, especially focusing on postmortem aging, using capillary-electrophoresis time-of-flight mass spectrometry (CE-TOFMS) metabolomics targeting water-soluble metabolites. The metabolomic data of JBR cattle were subjected to bioinformatic analyses, including metabolite pathway analysis. The data were compared to a dataset of postmortem muscle aging in JBL cattle. Interbreed differences are discussed in this article.
DISCUSSION
In the present study, a total of 240 metabolites on HMD and/or KEGG database were observed across all the JBRT beef samples in CE-TOFMS metabolomics. These metabolome profiles are considerably abundant in water-soluble, metabolically functional metabolites that have hardly been observed so far. Owing to the large number of annotated metabolites, several pathways and metabolisms were newly extracted in the postmortem aging process of JBRT beef, such as pyruvate metabolism, acetyl group transfer into mitochondria, and mitochondrial β-oxidation. Furthermore, interbreed differences in aged beef metabolites and postmortem muscle metabolisms were determined between JBRT and JBL breeds.
In the LT muscle of JBRT cattle, changes in the levels of a variety of metabolites were observed during postmortem aging. As aging progressed, significant increases were observed in the levels of F6P, G6P, IMP, inosine, hypoxanthine, leucine, glutamate, isoleucine, nicotinamide, creatinine, and butyrylcarnitine. However, ATP, G3P, malic acid, NAD+, and GSSG levels decreased. Such changes accompanying aging were also apparent in NTPs, NDPs, F-1,6-diP, PRPP, and taurine which showed a decreasing trend, and CDP-choline, succinic acid, several AAs and peptides, thiamine, cytidine, uridine, 6-phosphogluconic acid (6-PG), ribose 5-phosphate (R5P), and ribulose 5-phosphate (Ru5P) which exhibited an increasing trend. These metabolites can be categorized into glycolytic products, purine compounds generated by ATP degradation, citrate cycle-related metabolites, protein degradation products, pentose phosphate pathway-related metabolites, and other metabolite groups. This suggests that metabolic pathways such as glycolysis, purine metabolism (especially ATP and GTP degradation), citrate cycle, protein degradation, and the pentose phosphate pathway progressed predominantly in the postmortem JBRT LT muscle. The AAs and peptides were likely generated by postmortem protein degradation rather than
de novo biosynthesis or interconversion between AAs, as shown by accumulated evidence of protein degradation during postmortem aging [
13,
14,
16,
17].
The progress of these metabolic pathways was re-emphasized by the MSEA results, which demonstrated that pyrimidine metabolism, nicotinate and nicotinamide metabolism, purine metabolism, pyruvate metabolism, the Warburg effect, thiamine metabolism, citrate cycle, and pentose phosphate metabolism were the major metabolic pathways in postmortem JBRT beef (
Figure 5). These metabolic pathways were also developed in postmortem JBL and porcine LT muscles [
12,
24,
25]. The AA-associated metabolism extracted in MSEA, based on the accumulation of AAs and peptides, could be due to protein degradation, as observed in the postmortem aging of bovine muscles [
12,
24]. Postmortem protein degradation in porcine and bovine muscles is a well-established event based on many previous studies on troponin-T and other proteins [
13,
14,
16,
17]. Taken together, it is concluded that a series of coordinated metabolic events during the postmortem aging of JBRT beef is a conserved metabolic event in the postmortem skeletal muscle. In addition, the MSEA results of our study showed that fatty acid metabolism and mitochondrial metabolism were also activated in JBRT beef. Mitochondrial β-oxidation, fatty acid metabolism, and glycerolipid metabolism can also progress during the aging period, as shown in previous liquid chromatography-mass spectrometry metabolomics and lipidomics studies [
26,
33]. The present metabolomics results revealed an increasing trend of sugar phosphates (F6P, G6P, R5P, Ru5P), IMP, AAs including glutamate and creatinine, peptides, and 6-PG in postmortem JBRT beef aging. As sugars and AAs generate the Maillard reaction products through cooking process [
10], these metabolite changes may contribute to flavor development in JBRT beef, together with increase in IMP, an important
umami flavor compound. In this beef, redox metabolisms including glutathione and nicotinamide-related pathways seemed to change during the postmortem aging, which suggests progress of oxidation in the JBRT beef accompanying alteration of mitochondrial metabolisms. This may lead to lipid oxidation and discoloration of beef.
In the present study, we annotated 240 metabolites, the number of which was greater in JBRT beef in the present study than in JBL beef (171 metabolites) in a previous study [
24]. This high annotation of metabolites in JBRT beef was largely due to the improved detection systems and metabolomic information in the database, compared to that of the previous study. Owing to this improvement, the bioinformatic analysis of JBRT beef using MSEA resulted in new findings on postmortem muscle metabolism, such as transfer of acetyl groups into mitochondria, riboflavin metabolism, mitochondrial electron transport chain, fatty acid metabolism, mitochondrial β-oxidation of long/medium chain saturated fatty acids, butyrate metabolism, and inositol (phosphate) metabolism (
Figure 5).
We further analyzed the potential interbreed differences in the metabolome profiles of JBRT and JBL beef, based on the absolutely quantified metabolite levels to avoid great case-sensitive variation in the metabolomics analysis. In the HCA of samples during postmortem aging, interbreed differences were partly observed in the temporal changes in AAs, NTP degradation products, polyamines, glycolytic products, and other metabolites (
Figure 6). However, in the interbreed comparison of D14 samples, a large variation in individual animal samples was observed in most of these metabolites, such as homoserine, spermine, ADP, AMP, carnosine, creatinine, GSSG, fumaric acid, and betaine in JBRT or JBL beef (
Figure 10). Nevertheless, the levels of GMP, IMP, UMP, and F-1,6-diP were greater in JBRT beef than in JBL beef at day 14 postmortem, whereas the levels of choline, S7P, G3P, glycine, and other AAs were lower (
Figure 8). Furthermore, according to PLS-DA results, JBRT and JBL beef samples could be discriminated by a set of these metabolites with high VIP scores. Accordingly, these metabolites could be useful as biomarkers for discriminating JBRT beef from JBL beef. Similarly, interbreed differences in muscle metabolites have been reported in bovine, porcine, and sheep muscles [
30], some of which can be associated with differences in meat quality. In particular, IMP, phosphorylated sugars, and AAs contribute to meat flavor in beef and pork [
34]. The differences in the metabolites between JBRT and JBL beef are likely associated with the differences in the flavor of each beef. Intriguingly, pH values at day 1 and 14 postmortem were different between the breeds in this study. This is likely associated with lower lactate concentration in JBRT beef than in JBL beef at day 1 and 14 postmortem, suggesting lower glycolytic activity or lower glycogen content in JBRT. It is well established that the ultimate pH value affects meat quality such as IMP content [
12]. This may lead to higher umami flavor in JBRT beef than in JBL beef.
In addition, metabolites with interbreed differences can be generated through differently activated metabolism. MSEA in our study indicated that aged beef from the two breeds are different in metabolism related to not only various AAs but also phosphatidylethanolamine, phospholipid, glutathione, carnitine, purine, and phosphatidylcholine (
Figure 12). This difference in purine metabolism could likely contribute to the higher accumulation of IMP in JBRT beef than in JBL beef. The lower accumulation of AAs in JBRT beef might be due to the lower activities of postmortem proteolysis than in JBL beef. Furthermore, the interbreed difference in carnitine metabolism may indicate that the beef of the two breeds differed in mitochondrial metabolism associated with fatty acid transportation and oxidation, which could further affect the redox state and glutathione levels in myofiber cells. The different fatty acid metabolic processes may also be linked to differences in intramuscular fat accumulation between the breeds. These differences may contribute to interbreed difference in flavor, tenderness, and meat color. Further investigation by sensory evaluation is required to understand the interbreed differences in meat quality between JBRT and JBL breed. Currently, little is known about the genetics, genomics, mRNA, and protein expression in JBRT skeletal muscle. Further studies are required to investigate mechanism underlying interbreed differences by analyzing the genetic background and effect of postmortem aging conditions on JBRT beef.
CONCLUSION
In the LT muscle of JBRT cattle, metabolism related to purine/pyrimidine, nicotinate and nicotinamide, pyruvate, thiamine, amino sugar, fatty acid, butyrate, inositol, glycerolipid and mitochondrial fatty acid; and the citric acid cycle and pentose phosphate pathway were prominent pathways during the postmortem aging process. Of these, glycolysis, AA/peptide generation, and metabolism related to purine/pyrimidine, nicotinate/nicotinamide, pyruvate, and thiamine were the most common between JBRT and JBL breeds. Accumulation of IMP, UMP, and F-1,6-diP was greater, but that of choline, S7P, G3P, and various AAs was lower in aged JBRT beef than in JBL beef, indicating usefulness of these metabolites as potential biomarker candidates for discriminating between JBRT and JBL beef. The interbreed difference in beef metabolites could be due to metabolic differences related to AAs, phosphatidylethanolamine, phospholipid, glutathione, carnitine, purine, and phosphatidylcholine.